2.

2 Dilution Bottles or Tubes Use bottles or tubes of resistant glass, preferably borosilicate glass, closed with glass stoppers or screw caps equipped with liners that do not produce toxic or bacteriostatic compounds on sterilization. Do not use cotton plugs as closures. Mark graduation levels indelibly on side of dilution bottle or tube. Plastic bottles of nontoxic material and acceptable size may be substituted for glass provided that they can be sterilized properly. 2.3 Pipets and graduated cylinders Use pipets of any convenient size, provided that they deliver the required volume accurately and quickly. The error of calibration for a given manufacturers lot must not exceed 2.5%. Use pipets having graduations distinctly marked and with unbroken tips. Bacteriological transfer pipets or pipets conforming to APHA standar may be used. Do not pipet by mouth; use a pipet aid. Use graduated cylinders meeting ASTM Standards (D-86 and D-126) and with accuracy limits established by NITS where appropriate. Before sterilization, loosely cover opening of graduated cylinders with metal foil or a suitable heavy wrappingpaper substitute. Immediately after sterilization secure cover to prevent contamination. 2.4 Containers for culture medium Use clean borosilicate glass flasks. Any size or shape of flask may be used, but erlenmeyer flasks with metal caps, metal foil covers, or screw caps provide for adequate mixing of the medium containded and are convenient for storage 2.5 Culture dishes Use sterile borosilicate glass o disposable, presterilized plastic petri dishes, 60x15mm, 50x9mm, or other appropriate size. Wrap convenient numbers of clean, glass culture dishes in metal foil if sterilized by dry heat, or suitable heavy wrapping paper when autoclaved. Incubate loose-lidded glass and disposable plastic culture dishes in tightly closed containers with wet paper or cloth to prevent moisture evaporation with resultant drying of medium and to maintain a humid environment for optimum colony development. Presterilized disposable plastic dishes with tight-fitting lids that meet the specifications above are available commercially and are used widely. Reseal opened packages of disposable dish supplies for storage. 2.6 Filtration units The filter-holding assembly (constructed of glass, autoclavable plastic, porcelain, or stainless steel) consists of a seamless funnel fastened to a base by a locking device or by magnetic force. The design should permit the membrane filter to be held securely on the porous plate of the receptacle without mechanical damage and allow all fluid to pass through the membrane during filtration. Discard plastic funnels with chipped surfaces. Wrap the assembly (as whole or separate parts) in heavy wrapping paper or aluminum foil, sterilize by autoclaving, and store until use. Alternatively expose all surfaces of the previously cleaned assembly to ultraviolet radiation (2 min exposure) for the initial sanitization before use in the test procedure, or before reusing units between successive filtration series. Field units may be sanitized by dipping or spraying with

alcohol and then igniting or immersing in boiling water for 2 min. After submerging unit in boiling water, cool it to room temperature before reuse. Do not ignite plastic parts. Sterile, disposable field units may be used. For filtration, mount receptacle of filter-holding assembly on a 1-L filtering flask with a side tube or other suitable device (manifold to hold three to six filter assemblies) such that a pressure differential (34 to 51 kPa) con be exerted on the filter membrane. Connect flask to a vacuum line, an electric vacuum pump, a filter pump operating on water pressure, a hand aspirator, or other means of securing a pressure differential (138 to 207 kPa). Connect a flask of approximately the same capacity between filtering flask and vacuum source to trap carry-over water. 2.7 Membrane filter Use membrane filters with rated pore diameter such that there is complete retention of bacteria. Use only those filter membranes that have been found, through adequate quality control testing and certification by the manufacturer, to exhibit: full retention of the organisms to be cultivated, stability in use, freedom from chemical extractables that may inhibits bacterial growth and development, a satisfactory speed or filtration (within 5 min), no significant influence on medium pH (beyond +/- 0.2 units), and no increase in number of confluent colonies or spreaders compared to control membrane filter. Use membranes grid-marked in such a manner that bacterial growth is neither inhibited nor stimulated along the grid lines when the membranes with entrapped bacteria are incubated on a suitable medium. Preferably use fresh stocks of membrane filters and if necessary store them in an environment without extremes of temperature and humidity. Obtain no more than a years supply at any one time. Preferably use presterilized membrane filters for which the manufacturer has certified that the sterilization technique has neither induced toxicity nor altered the chemical or physical properties of the membrane. If membranes are sterilized in the laboratory, autoclave